Researches On The New Mehods For Fluorescence Detection Of Pathogenic Microorganisms And Sludge Reduction By Biomimetic Enzyme Technology

With the growing problem of environmental pollution, pathogenic microorganisms are present in our lives everywhere. Because of neglected the seriousness and danger of pathogens, there are many infectious disease outbreak events all around the world. The traditional microbiological testing methods can not meet the needs of rapid and efficient testing of the large scale and paroxysmal disease. Therefore, it is an urgent need to develop an accurate, fast and efficient method for detecting pathogenic microorganisms in environment. In addition, it is the other serious and easy to ignore environmental problem that the sludge production from urban sewage treatment plant are increasing but not to be sufficient treatment and disposal. As we all know, sludge contains large amounts of pathogenic microorganisms, heavy metals, persistent organic compounds and other toxic substances, which will cause secondary pollution and pollute the groundwater, soil and atmosphere. And there will be a serious threat to human health and environmental safety. For this reason, many environmentalists have started to focus on the sludge issue of effective treatment and disposal.In our research group, we have an abundant foundation of fluorescence detection and also in the field of porphyrins and its derivatives have carry out a large number of basic research and applications, and made some research. And based on the above two major environmental issues and our advantage of research, this work will focus on rapid detection methods of pathogens and the reduction technology of excess sludge. The details are summarized as follows:(1) A two-probe tandem DNA hybridization assay based on time-resolved fluorescence analysis method was employed to rapid detect Escherichia coli strain. The results showed that the optimal concentration of capture probe immobilized on glass surface was1.0×10-7mol/L, and the optimal hybridization time and temperature were10h and43℃, respectively. The detection method has good specificity and sensitivity, and the method also successfully applied to the soil and the landscape in the detection of E. coli. Furthermore, without PCR amplification and DNA purification process, the extracted nucleic acid was directly used to the hybridization process, and a satisfactory specificity and sensitivity result was obtained, which shows that the method is easy to operate, simple and feasible, and economical. It can be concluded that this method is a promising technique for monitoring the microorganisms.(2) Based on the above method, it was re-established a fast and efficient way to detect Staphylococcus aureus. In this study, the hybridization temperature, hybridization time, washing time and other conditions were re-optimized. Results showed that the optimal hybridization time is9h, the optimal hybridization temperature at53℃,the best time for washing4.5min. The detection method has good specificity and sensitivity, and it also successfully detected a simulated contaminated fruit juice and milk in the detection of Staphylococcus aureus.(3) Three kinds of core-shell nanoparticles were successfully synthesized by the microemulsion method. Among these nanoparticles, the one of europium complex Eu(TTA)3(5-NH2-phen) with IgG-modified has satisfactory fluorescence performance and package stability. The nanoparticle was labeled with DNA probes and employed to the hybridization reaction of target nucleic acid from the Escherichia coli and Staphylococcus aureus, respectively. Results showed that the fluorescence intensity of the two hybrid systems were slightly increased, but the reproducible performance of this detection systems were not better than that of the molecular europium complex. It may be due to agglomeration between nanoparticles. So, if improving the dispersion property of the nanoparticles in aqueous, it will be a very promising biomarker in the field of molecular biology.(4) A fluorescence resonance energy transfer based competitive hybridization of nucleic acid detection methods was presented, in which naphthalimide EEN (N-hydroxyethyl4-(2-amino ethane) amino-1,8-naphth-alimide) was the donor dye and anthocyanin dye Cy5was the receptor substances. A series of Acceptor probe-1concentrations were employed to hybrid with Donor probe connected the EEN dyes. A satisfactory result of fluorescence resonance energy transfer was obtained. In the nucleic acid competition hybridization procedure, the Acceptor probe-1was effectively replaced by Target probe, which was appeared that the fluorescence intensity of EEN was restored and that of Cy5was weaken. In conclusion, it has successfully established fluorescence resonance energy transfer-based competitive hybridization of nucleic acid detection methods.(5) The metal iron porphyrin Fe(TAPP)Cl synthesized by our team, as a biomimetic enzyme cytochrome P450, was applied to the excess sludge anaerobic digestion process. Results showed that the iron porphyrin can be immobilized on the surface of ordinary glass beads, and they worked effectively on the sludge digestion. When used the ethanol solution of iron porphyrin as biomimetic cytochrome monooxygenase in the sludge digestion process, the optimum digestion time was7h, the optimal treated temperature at40℃and the best percentage of dosage is about1%. It is apparent that this method has the advantages of easy operation, mild reaction condition and economical equipment. Therefore, compared with the natural enzymes, the chemical synthesis of biomimetic enzyme has the superiority in the operation condition and application scope.